CN107137701B - 一种提高dc疫苗抗肝癌效果的基因靶标、抑制剂和dc肿瘤疫苗 - Google Patents
一种提高dc疫苗抗肝癌效果的基因靶标、抑制剂和dc肿瘤疫苗 Download PDFInfo
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Abstract
本发明公开了一种提高DC疫苗抗肝癌效果的基因靶标、抑制剂和DC肿瘤疫苗。本发明证明，通过siRNA抑制DC中SOCS2表达可以显著提高DC疫苗的体内抑瘤效果，而本发明提供的外源肽为DC中SOCS2表达的有效抑制剂，具有与siRNA相似的功能，可以显著提高DC疫苗的体内抑瘤效果。使用SOCS2的表达抑制剂和本发明提供的外源肽可以用于制备更优抗肝癌效果的DC肝癌疫苗。
Description
技术领域
本发明属于基因治疗和免疫治疗领域，具体涉及一种提高DC疫苗抗肝癌效果的基因靶标、抑制剂和DC肿瘤疫苗。
背景技术
肝细胞癌是全世界最常见的恶性肿瘤之一，每年约有60万患者被诊断出患有肝细胞癌。目前，对于肝细胞癌的治疗主要是手术切除、肝脏移植、放化疗等，但仅有20％的患者能够被治疗。我国肝细胞癌年死亡率占肿瘤死亡率的第2位。肝细胞癌的病因及发病机制尚未确定，因此，寻找新的肝细胞癌的治疗靶点和药物是研究重点。
基于DC的生物治疗格外引人瞩目。理论上说，DC是激发适应性免疫最有效的APC，将其在体外荷载肿瘤抗原后回输，荷肽DC通过MHC/抗原肽-TCR的相互作用激活特异性T细胞，从而启动抗瘤免疫应答，预防或者清除肿瘤。这种方法安全而少副作用，对微小和转移病灶更有传统手段无可比拟的高度靶向性。但实际运用时，仅仅荷肽的DC往往并不能激发足够强大的抗瘤免疫应答，所以治疗效果不如预期理想。这是由于为躲避宿主免疫系统攻击，肿瘤发展出多种多样的逃逸机制，在抗原提呈阶段，就可能存在肿瘤抗原免疫原性低下、MHC分子表达减少、协同刺激分子缺如、分泌抑制性细胞因子等因素。因此，为提高疫苗的效能，就必须采取一定的方法来修饰、处理DC，增强其抗原提呈能力。
发明内容
本发明目的在于提供一种提高DC疫苗抗肝癌效果的基因靶标、抑制剂和DC肿瘤疫苗。
实现本发明上述目的技术方案如下：
基因SOCS2作为靶标在制备DC肝癌疫苗方面的应用。
基因SOCS2的抑制剂在制备DC肝癌疫苗方面的应用。
优选地，所述抑制剂为抑制基因SOCS2表达的siRNA或含抑制基因SOCS2表达的siRNA序列的载体。
优选地，所述抑制剂为SEQ ID NO.1所示的外源肽。
优选地，所述外源肽N和/或C末端被化学修饰。
优选地，所述外源肽的N末端被乙酰化修饰，C末端被酰胺化修饰。
优选地，所述外源肽为游离形式或药学上可以接受的成盐形式，包括盐酸盐、硫酸盐、磷酸盐、磺酸盐、乙酸盐、柠檬酸盐和酒石酸盐。
一种生物制品，含有上述抑制剂。
上述生物制品在制备DC肝癌疫苗方面的应用。
一种DC肝癌疫苗，其DC细胞的SOCS2基因低表达。
本发明的突出优点：
本发明证明，通过siRNA抑制DC中SOCS2表达可以显著提高DC疫苗的体内抑瘤效果，而本发明提供的外源肽为DC中SOCS2表达的有效抑制剂，具有与siRNA相似的功能，可以显著提高DC疫苗的体内抑瘤效果。使用SOCS2的表达抑制剂和本发明提供的外源肽可以用于制备更优抗肝癌效果的DC肝癌疫苗。
附图说明
图1为各组成熟DC中SOCS2 mRNA的相对表达量；
图2为各组成熟DC接种20天后对荷瘤裸鼠的体内抑瘤率(％)。
具体实施方式
下面就结合实施例具体介绍本发明的实质性内容，由于篇幅原因，实验过程的描述无法做到非常详细，凡是实验中未详细描述的部分均为本领域技术人员熟知的常规操作。
一、实验材料
人肝细胞株瘤细胞株HHCC购于美国ATCC，并有本公司保存。将HHCC细胞复苏后接种于培养瓶中，加入含有10％FBS的RPMI-1640，贴壁细胞用0.25％的胰酶消化。取对数生长期的细胞进行实验。Balb/c小鼠购于南京君科生物工程有限公司。
本发明外源肽通过本领域公知的标准多肽固相合成技术合成，采用芴甲氧羰基(Fmoc)N端保护策略。按照树脂固相合成的方法依次连接相应氨基酸，期间依次脱去Fmoc-保护基团，然后切肽，获得粗品，粗品经C18柱分离纯化，HPLC和MS检测确证了本发明外源肽的结构，且纯度均≥98％。其氨基酸序列如下(N末端→C末端)，末端未经化学修饰：
QEDLMGPNQTDGEPC(SEQ ID NO.1)
二、实验方法
1、细胞培养和肿瘤裂解物的制备
人肝细胞株瘤细胞株HHCC购于美国ATCC，并由本公司保存。将HHCC细胞复苏后接种于培养瓶中，加入含有10％FBS的RPMI-1640，贴壁细胞用0.25％的胰酶消化。取对数生长期的细胞进行实验。Balb/c小鼠购于南京君科生物工程有限公司。
肿瘤裂解物的制备：将HHCC细胞接种于含有10％FBS的RPMI-1640的培养瓶中，待贴壁达到80％时，取贴壁细胞，用PBS洗涤重悬至3×107/mL，经-80℃/37℃反复冻融5次，超声波裂解1次，离心收集上清即得肿瘤裂解物，冷冻干燥。
2、BM-DC的培养
参考Garrigan等人方法：RPMI-1640冲洗小鼠长骨髓腔获得骨髓细胞，Tirs-NH4裂解去除红细胞，用含10％FCS、20ng/ml rmGM-CSF(R&D)的RPMI-1640完全培养基重悬至3×106细胞/ml，1ml/孔接种至24孔培养板，37℃、5％CO2培养。每隔2-3d，轻轻吹打，3/4量换液以去除悬浮细胞，并补液至原体积。5～7d后，收集悬浮和松散贴壁的细胞即为未成熟的树突状细胞(iDC)。
3、外源肽和siRNA沉默SOCS2基因表达
siRNA沉默组：收集培养第6天的iDC，转染有绿色荧光染料FAM标记、针对SOCS2mRNA的siRNA(委托上海吉玛设计合成)，步骤如下：按每孔3μl FAM-siRNA、3μlGeneporter(Gene Therapy Systems)、96μl无血清RPMI-1640混合，接种至12孔培养板，25℃孵育30min。随后，用无血清RPMI-1640重悬iDC至2×106细胞/ml，以1ml/孔加入孵育有转染试剂的12孔培养板，37℃孵育4h；最后，每孔加入1ml含10％FCS、20ng/ml rmGM-CSF的RPMI-1640完全培养基，37℃孵育8h。
外源肽沉默组：收集培养第6天的iDC，不转染siRNA，于12孔培养板中以含10％FCS、20ng/ml rmGM-CSF的RPMI-1640完全培养基调整成与siRNA沉默组终体积相同、细胞浓度相同的培养体系，但是培养基中含终浓度为10μM的外源肽，37℃孵育8h。
阴性对照组：同外源肽沉默组，但是培养基中不含外源肽，37℃孵育8h。
4、DC荷载肿瘤裂解物与LPS刺激成熟
未沉默和沉默SOCS2的iDC用含肿瘤裂解物(200μg/ml))、LPS(100ng/ml)的RPMI-1640完全培养基重悬至2×106细胞/ml，1ml/孔接种至24孔板，37℃孵育24h以成熟。其中，未沉默SOCS2者标记为NSI-DC，siRNA沉默者标记为SSI-DC，外源肽沉默者标记为PSI-DC。通过流式法检测检测CD11c、CD80、CD86的表达检测DC纯度和成熟度。
5、RT-PCR检测DC中SOCS2 mRNA表达
用Trizol法提取NSI-DC、SSI-DC、PSI-DC的总RNA。采用两步法RT-PCR，参照TaKaRa公司AMV反转录酶说明书进行反转录。cDNA的PCR反应体系如下：2μL反转录产物，5μL的10×PCR Buffer(Mg2+Free)，3μL MgCl2(25mmol/l)，4μL的dNTP Mixture(2.5mmol/L)，0.25μL的Taq酶(5U/μl)，上下引物各1μL(l20μmol/l)，加灭菌蒸馏水调至50μl体系。选用小鼠GAPDH作内参，引物序列如下：
SOCS2上游引物：5’-AGCTCGGTCAGACAGGATGG-3’(SEQ ID NO.2)
SOCS2下游引物：5’-TTCGAAGATTAGTTGGTCCAGC-3’(SEQ ID NO.3)
GAPDH上游引物：5’-ATATGGTACGCGAGGTGAGG-3’(SEQ ID NO.4)
GAPDH下游引物：5’-GTGATTTTAGGGACGCTCCA-3’(SEQ ID NO.5)
PCR产物在1.5％琼脂糖凝胶上电泳，GV染色后紫外线投射仪下观察结果，经凝胶扫描成像系统拍照，用Quantityone软件半定量分析结果，即SOCS2 mRNA条带的灰度值/GAPDH条带的灰度值，取平均值进行统计学分析。
7、荷瘤动物的建模与治疗
Balb/c小鼠于右背部皮下接种HHCC细胞1×106/只，接种2周后成瘤率100％，瘤径约5mm。将成瘤裸鼠随机分为4组：siRNA沉默组、外源肽沉默组、阴性对照组和空白对照组，每组10只。从第15天开始，siRNA沉默组、外源肽沉默组、阴性对照组每2天分别于皮下注射SSI-DC、PSI-DC、NSI-DC的灭菌PBS悬液(1×106/50μL)，空白对照组注射等量灭菌PBS，连续注射4次。为了充分促进机体免疫反应，每次注射DC或PBS后再腹腔注射LPS，每只每次30μg/200μL。第25天开始(接种DC第10天)，游标卡尺每2天测一次肿瘤体积。接种DC 20天后，处死小鼠，剥离肿瘤块，计算siRNA沉默组、外源肽沉默组、阴性对照组相对于空白对照组的肿瘤抑制率(％)，公式：肿瘤抑制率(％)＝(空白对照组体积-siRNA沉默组/外源肽沉默组/阴性对照组体积)/空白对照组体积×100％。
8、数据处理
数据分析采用SPSS20.0软件进行，数据表示方法为均值±标准差，组间比较采用t检验，P<0.05认为有统计学差异。
三、实验结果
1、各组DC纯度和成熟度
CD11c是小鼠髓系来源DC的特异性标志，各组DC的CD11c均呈阳性表达，且纯度在80％以上。CD80、CD86是与DC抗原呈递相关的标志分子，成熟前均低表达，但成熟后均显著高表达，其中，成熟DC的CD80的纯度均在80％以上，CD86的纯度均在50％以上。
结果表明，NSI-DC、SSI-DC、PSI-DC均为成熟的DC细胞。
2、各组成熟DC中SOCS2 mRNA表达
与阴性对照组NSI-DC相比，siRNA沉默组SSI-DC、外源肽沉默组PSI-DC中的SOCS2mRNA表达显著下调(P<0.05)，各组SOCS2 mRNA相对表达量见表1和图1。
表1各组成熟DC中SOCS2 mRNA的相对表达量
| 组别 | SOCS2 mRNA相对表达量 |
| 阴性对照组 | 1.24 |
| siRNA沉默组 | 0.32 |
| 外源肽沉默组 | 0.39 |
3、各组成熟DC对荷瘤裸鼠的体内抑瘤率(％)
与阴性对照组NSI-DC相比，siRNA沉默组SSI-DC、外源肽沉默组PSI-DC对荷瘤裸鼠的体内抑瘤率显著升高，肿瘤生长缓慢，具有明显的抑制作用。各组成熟DC接种20天后对荷瘤裸鼠的体内抑瘤率(％)见表2和图2。
表2各组成熟DC接种20天后对荷瘤裸鼠的体内抑瘤率(％)
| 组别 | 体内抑瘤率(％) |
| 阴性对照组 | 35 |
| siRNA沉默组 | 73 |
| 外源肽沉默组 | 67 |
实验证明，通过siRNA抑制DC中SOCS2表达可以显著提高DC疫苗的体内抑瘤效果，而本发明提供的外源肽为DC中SOCS2表达的有效抑制剂，具有与siRNA相似的功能，可以显著提高DC疫苗的体内抑瘤效果。使用SOCS2的表达抑制剂和本发明提供的外源肽可以用于制备更优抗肝癌效果的DC肝癌疫苗。
上述实施例是对本发明实质性内容的体现，用于更好地解释本发明，但本领域技术人员应当知晓，不应将本发明的保护范围局限于上述具体的实施例。
SEQUENCE LISTING
<110> 南京盖斯夫医药科技有限公司
<120> 一种提高DC疫苗抗肝癌效果的基因靶标、抑制剂和DC肿瘤疫苗
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<160> 5
<170> PatentIn version 3.3
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Claims (1)
1.一种基因SOCS2的抑制剂在制备DC肝癌疫苗方面的应用，其特征在于：该基因SOCS2的抑制剂为SEQ ID NO. 1所示的外源肽。
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